A pilot small molecule siRNA screen was performed for inhibitors of mTORC1 signaling. Replicability of In-Cell Western Assay for siRNA screening via a correlation plot. B) Signal intensities from In-Cell Western wells are plotted as a proportion of the mean values for each of two plates (plate 1 in blue, plate 2 in red). Band intensities are expressed as a proportion of the mean of the thirteen samples on each blot (blot 1 in blue, blot 2 in red). A) GAPDH and PMLC20 were detected on duplicate Western blots (13 replicates per blot, not shown). Intra-assay variability of Western blots and In-Cell Western Assays. In-Cell Western Assays offered superior precision, reduced variability, and smaller CVs. In-Cell Western Assay and Western blot analysis yielded very similar results (Figure 1). 1 Primary cultures of uterine myocytes stimulated with oxytocin were used to assess specificity, sensitivity, and precision of the two methods for phospho-analysis. 2, 4, 5Ī 2010 study compared In-Cell Western Assays and Western blotting (WB) for measurement of phosphorylated myosin regulatory light chain (PMLC20). If they overlap, or nearly overlap (Z′ 0.5). Z′ is calculated by running a large number of positive and negative controls and determining how much separation there is between positive and negative controls. Z′-Factor can provide some indication of the replicability of an assay. Z′-Factor is a measure of statistical effect size and can be used to assess if a response to an assay requires further investigation. Produces excellent Z′-Factor with optimized conditions and experimental design.Very similar profiles of signal increase and decrease when compared to Westerns.Characterize a broad range of cell signaling parameters.Easily run many replicates to increase accuracy (Figure 2).Replicate measurements with very low coefficients of variation (CVs) (Figure 1).Significantly smaller standard deviations.In-Cell Western Assays exhibit the following characteristics: In-Cell Western Assays provide greater replicability and precision than Western blots. Target Analysis: Quantify the effect of a treatment or condition on a target.Z′-Factor Determination: Test the quality and robustness of an assay.Fixation and Permeabilization Evaluation: Determine the optimal fixation and permeabilization conditions for an experiment.Blocker Evaluation: Determine the best blocking buffer or blocking buffer and antibody combination for an experiment.Antibody Titration: Determine the antibody concentration that provides optimal signal and lowest background.Cell Stain Linearity: Ensure signal intensity for the normalization method and target are each detected within their linear range.For In-Cell Western Assays, create a custom template or use one of the six preset templates for: These straightforward and systematic workflows in Empiria Studio automate the critical steps of your analysis.
#Dot blot 96 wells software#
Normalize to cell number, allowing for accurate quantification and comparison of protein expression between wellsĮmpiria Studio ® Software has step-by-step workflows and Plate Templates for various types of multiwell plate assays, including the In-Cell Western Assay.Detect proteins in situ in a relevant cellular context.Quickly, accurately measure relative protein levels in many samples.Quantify multiple targets using spectrally distinct fluorescent dye conjugates.Detect proteins in fixed and permeabilized cells using target-specific primary antibodies and IRDye ® Secondary Antibodies.In-Cell Western Assays are also called cytoblots, cell-based ELISA, In-Cell ELISA (ICE), and FACE (Fast Activated Cell-based ELISA). The In-Cell Western Assay is a quantitative immunofluorescence assay performed in multiwell plates (optimized for 96- or 384-well format) that combines the specificity of Western blotting with the replicability and throughput of ELISA.
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